Antibiotic s 15-1 and process for producing the same

ABSTRACT

NEW ANTIBIOTIC S 15-1, C18H36N6O10, WHICH HAS WIDE ANTIM,ICROBIAL POWER AGAINST GRAM-POSIITIVE, GRAM-NAGATIVE, ACID FAST BACTERIA AND, PARTICULARLY, NEWCASTLE DISEASE VIRUS AND FURTHER, WHEN IN HIGH CONCENTRATION, AGAINST MOLDS AND YEASTS IS PRODUCED BY CULTURING AEROBICALLY ST. GRISEOCARNEUS S 15-1 NRRL 5311 IN A CULTURE MEDIUM CONTAINING CARBON AND NITROGEN SOURCES AND OTHER NUTRITIVE MATERIALS UNTIL ANTIBIOTIC S 15-1 IS ACCUMULATED AND THEN RECOVERING THE ANTIBIOTIC FROM THE MEDIUM.

June 4, 1974 V g ARIMA ETAL 3,814,795

ANTIBIO'I'IC S15 1 AND PROCESS FOR PRODUCING THE SAME Filed Nov. 15, 1972 220 K 240 \zo 3 200 300 230 250 270 290 WAVE LENGTH (mp) FIG. 2

I I I I I I I I I I I 3600 3200 2800 2400 2000 I800 I600 I400 I200 I000 800 United States Patent 3,814,795 ANTIBIOTIC S 15-1 AND PROCESS FOR PRODUCING THE SAME Kei Arima, Tokyo, Takeshi Kawamnra, Saitama, Gakuzo Tamura, Tokyo, and Teruhiko Beppu, Matsudo, Japan, assignors to Asahi Kasei Kogyo Kabushiki Kaisha, Osaka, Japan Continuation-impart of abandoned application Ser. No. 177,382, Sept. 2, 1971. This application Nov. 15, 1972, Ser. No. 306,878

Claims priority, application Japan, Sept. 7, 1970, 45/ 77,803 Int. Cl. A61k 21/00 U.S. Cl. 424-116 ABSTRACT OF THE DISCLOSURE New antibiotic S 15-1, C H N O which has wide antimicrobial power against gram-positive, gram-negative, acid fast bacteria and, particularly, Newcastle disease virus and further, when in high concentration, against molds and yeasts is produced by culturing aerobically St. griseocameus S 15-1 NRRL 5311 in a culture medium containing carbon and nitrogen sources and other nutritive materials until antibiotic S 15-1 is accumulated and then recovering the antibiotic from the medium.

3 Claims 1 This application is a continuation-in-part application of our prior US. application Ser. No. 177,125,382 filed on Sept. 2, 1971, now abandoned.

This invention relates to a new antibiotic named as S 15-1 and a process for producing the same by culturing an antibiotic S 15-1 producing strain belonging to the genus Streptomyces and then recovering the resulting antibiotic S 15-1 from the culture.

Antibiotic S 15-1 is a new antibiotic having properties set forth hereinbelow and inhibits the growth of grampositive, grain-negative, and acid fast bacteria. When the antibiotic is used at a high concentration, it inhibits the growth of molds, yeasts and the like fungi. Furthermore, the antibiotic inhibits the growth in tissue culture of 'virus such as Newcastle disease virus. The antibiotic S 15-1 is useful in poultry husbandry.

' An object of this invention is to provide a new antibiotic S 15-1.

Another object of this invention is to provide a method for producing the new antibiotic S 15-1.

Other objects and advantages of this invention will be apparent from the description set forth hereinbelow.

The microorganism used in the present invention is a strain of the genus Streptomyces which can produce in the culture thereof the antibiotic S 15-1 in an amount suflicient to be recovered. As such strain of the genus Streptomyces, there may be shown, for example, a microorganism which is first isolated by the inventors from soil in the suburbs of Sendai City, Miyagi Prefecture, Japan. This microorganism is an S 15-1 which is identified by the inventors to belong to Streptomyces griseocarneus. The Streptomyces griseocarneus S 15-1 strain has such mycological properties as mentioned below. This strain is being deposited in Fermentation Research Institute, Agency of Industrial Science and Technology, Chiba City, Japan under FERM-P No. 706. This strain is also deposited with the United State Department of Agriculture, Northern Regional Research Laboratory, at

Peoria, Illinois, United States of America and has been given the number NRRL 5311; a sample of this microorganism can be obtained from aforesaid Research Laboratory. (The deposit was made with aforementioned Research Laboratories with all restrictions on the availability to the public being irrevocably removed upon the granting of the patent.)

I. MORPHOLOGICAL CHARACTERISTICS Growth in media with hyphae, as seen in actinomycets in general. Aerial mycelium, long and fibrous, or abundantly branched, sometimes slightly hooky; no whorls and no spirals. Ten or more conidia in chains at the tip of aerial mycelium, ellipsoidal or cylindrical, 0.6 to 0.7 by 1.1 to 1.3 microns. Spores, smooth surface.

II. BEHAVIORS ON VARIOUS CULTURE (1) Czapeks Dox agar plate (27 C.):

Growth cream-colored; aerial mycelium scant, white;

no soluble pigment.

(2) Glucose-asparagine agar plate (27 C.):

Colorless growth; aerial mycelium white, powdery,

scant. No soluble pigment.

(3) Starch synthetic agar plate (27 C.):

Colorless growth; white to ash-gray aerial mycelium, well developed. Gray soluble pigment, scant. Starch is actively hydrolyzed.

(4) Ca-malate agar plate (27 C.):

Colorless growth; aerial mycelium scant, white. No

soluble pigment.

(5) 0.2% Sodium nitrate-incorporated peptone water Colorless growth in masses at the bottom. No soluble pigment. Nitrites produced from nitrates. (6) Broth agar slant (27 C.):

Growth grayish yellow, wrinkled; no aerial mycelium.

Pale brown soluble pigment.

(7) Sugar-incorporated broth agar slant (27 C.):

Growth grayish yellow, wrinkled; aerial mycelium scant, white. Pale brown soluble pigment.

(8) Coagulated Loefllers blood serum slant (27 C.):

Growth white to ash-gray; no aerial mycelium.

Soluble pigment gray to grayish black. 1% liquefaction. No growth at 37.

(9) Bennetts agar plate (27 C.):

Growth yellow; thick aerial mycelium, pinkish gray.

Pale brown soluble pigment. No growth at 37 C. (10) Gelatin 12 C.): i [Growth colorless to pale brown; white, thick aerial mycelium in stab. Slow liquefaction. Pale brown soluble pigment. (11) Milk (30 C.):

Growth cream-colored to brown rings; no aerial mycelium. Pale brown soluble pigment. Becomes transparent from the upper layer portion, and has 3 III. BIOLOGICAL PROPERTIES Tyrosinase formation reaction Positive. Nitrite formation reaction Do. Skim milk coagulation reaction Negative. Skim milk peptonization reaction Positive (weak). Starch hydrolysis Positive (strong). Gelatin liquefaction reaction Positive (weak). Coagulated Loefiiers blood serum solubility Negative.

1v. UTILIZABILITY OF CARBON SOURCES Note:

-+: Utilized Not utilized Utilizability doubtful I l I I r I 3,814,79

rays, radioactive rays or chemicals. All the thus obtained variant's'm'ay also be used in the present process sofar As mentioned above, the S 15-1 strain forms an aerial mycelium having no whorls or spirals and produces smooth conidia, and is a strain of the chromogenic type which gives an aerial mycelium of white to pale red color, later becoming ash-gray. It shows a cream-colored growth on Czapeks agar, and a colorless growth on glucose-asparagine agar, starch synthetic medium or Camalate agar, and is broad in scope of carbon source utilization spectrum.

In view of such characteristics of the S 15-1 strain, there may be shown as homologous strains Streptomyces cinnamonensis, Streptomyces bikiniensis, Streptomyces antibioticus and Streptomyces griseocarneus.

Among these strains, Streptomyces cilmamonensis is distinguished from the S 15-1 strain in that the former develops a salmon pink-colored aerial mycelium on glucose-asparagine agar, whereas the latter shows a colorless growth. Streptomyces bikiniensis is distinguished from the S 15-1 strain in that it produces an amber-colored pigment on glucose-asparagine agar and a pale brown pigment on a synthetic medium. Streptomyces antibioticus is distinguished from the S 15-1 strain in that it shows a white growth on a synthetic medium, develops a greenish gray aerial mycelium on a milk medium, and forms a bluishpigment andshows a brown growth on a potato medium. The known Streptomyces griseocarneus more or less ditfers from the S 15-1 strain in nitrate reducibility, saccharide assimilability, etc., but Well coincides therewith in' other points. From the above resultsQthe inventors conclude that 'the'S 15-1 strain belongs to Strep tomyces griesocarneus, and distinguish the S 15-1'strain from the known strain, denominating the same as Streptomyces griseocarneus S 15-1. V f

The-properties of 'Streptomyces grz'seocarneus S '15-1 are as mentionedpreviously. However, the properties of this strain are liable to be varied as seen. in other strains of the genus Streptomyces, and are-variable by artificial means using, for example, ultiraviolet rays, X-

as they belong to Streptomyces griseocarneus and have ability to produce the antibiotic S 15-1.

In the present invention, the aforesaid strain is cultured in a medium containing nutrients capable of being utilized by known microorganisms. That is, the strain having antibiotic S 15-1 producing ability may be aerobically cultured in a medium containing a carbon source, a nitrogen source, inorganic salts and, if necessary, a slight amount of an organic nutrient source. As the nutrient sources, there may be used known sources which have heretofore been utilized for the culture of strains of the genus Streptomyces. For example, glucose, starch, dextrin, glycerin or the like may be used as the carbon source, and soybean flour, meat extract, peptone, corn steep liquor, ammonium sulfate, sodium nitrate or the like may be used as the nitrogen source. In addition thereto, there may be added, if necessary, inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, phosphate, etc., and a minor amount of such organic and inorganic materials as accelerating the growth of the strain and promoting the production of the antibiotic S 15-1.

As the culture method, a liquid culture method, particularly a submerged tank" culture method, is the most suitable, like in the case of production of known antibiotics. The culture is effected under aerobic conditions, and a temperature preferable for the culture is 20 to 35 C. pH for the culture is preferably from 5 to 9. In most cases, however, the culture is carried out at about 28 C. Thus, the amount of antibiotic S 15-1 produced reaches maximum in 2 to 6 days either in shaking culture or tank culture.

Antibiotic S 15-1 exhibits such physicochemical proper-'- ties as mentioned later, and hence can be extracted and purified according to the said properties. Antibiotic S 15-1 produced by the culture is recovered from a culture filtrate separated by filtration, either in the form of a base, in the form of a salt, in the form of an impure product or in the form of a purified solid. For example, antibiotic S 15-1 is recovered in such a manner as mentioned below.

The culture filtrate (beer) is adsorbed on a cation-exchange resin such as Amberlite IRC-50 (H-form), washed with water, eluted with a dilute acid, freed from excess acid by use of Amberlite IR-45 (OH-form), concentrated and then freeze-dried. The resulting crude powder is extracted with methanol, and the extract is concentrated and charged into a large amount of acetone to obtain anti-' biotic S 15-1 (hydrochloride) as a precipitate. This hydrochloride is subjected to cellulose column chromatography (eluted with an acetic acid-containing solvent), and the active portion is concentrated and then freeze-driedtoobtain a powder (acetate). The thus obtained powder is con verted into a hydrochloride or sulfate by use of an ion exchanged resin (H-form). When the above-mentioned acetate is treated with a weakly basic ion-exchange resin, theantibiotic is isolated as a free base. The hydrochloride, base and sulfate obtained in the above manner are white powders. 1 I

. Physicochemical properties of antibiotic S 15-1 are set forth below.

This substance (free base) decomposes at 161to 162 C. to develop 'a yellow color. Hydrochloride of the free base is well soluble in water and methanol, sparingly soluble in lower alkyl alcohols such as ethanol, and insoluble in such organic solvents as acetone, ethyl acetate, chloroform, ether, benzene and the like. The base and sulfate are well soluble in water, but sparingly soluble in methan01 and insoluble in other organic solvents. The optical rotation of the hydrochloride as measured in an aqueous solution thereof is (u) 26. The ultraviolet absorption curve of the antibiotic S 15-1 (hydrochloride) is shown in FIG. 1, and the infrared absorption curve (KBr tablet) is positive in each of ninhydrim'Fehlings, Elson-Morgans,

Molisch, silver mirror, anthrone, Sakaguchis, and maltol reactions, and negative in each of biuret and orcinol reactions. The results of elementary analysis of the base are as follows:

c: 431329;), 7.13%, N: 17.29% Molecular formula: C H N O Molecular weight: 496

"The R; values of antibiotics S l51 measured according to paper chromatography are as set forth in the following table:

Developer Aqueous saturated butanol 3% aqueous ammonium chloride solution 50% acetone water Phenohwater (3:1)-. n-Butanolzmethanolzwater (4:1:2) 6) Solvent"() (70 cc.) plus 1.5 g. of methyl orange (7) Benzenezmethanol 4: (8) Aqueous saturated bntanol plus 2% p-toluene sulfonic acid.

(9). n-Propanokpyridinezacetlc acidzwater In view;of the above-mentioned physicochemical properties, i.e. in view ofthe fact that the antibiotic S 15-1 is a water-soluble antibiotic having an optical rotation of and is maltol reaction positive, the antibiotics S 15-1 is regarded as an antibiotic of the Streptomycin type. As antibiotics of the Streptomycin type, there have been known streptomycin, dihydrostreptomycin, hydroxystreptomycin, mannosidostreptomycin and glebomycin. However, all these antibiotics are distinguished from the antibiotic S 15-1 in that they are different therefrom in optical rotation and are ninhydrin reaction negative.

Antimicrobial spectra of the antibiotic S 15-1 against various microorganisms are as shown in the following table:

Minimum inhibitory concentration Test microorganisms (meg/ml.) Medium Bacillus subtilis POI 219 0. 78 I Bacillus cereus IFO 3001 25. 0 1 Staphylococcus aureus 209-P. 0. 78 I Sarcina lutea POI 1001 6. 25 I Corynebacterium sum. 25. 0 I Escherichia coil K-12- 3. 13 I Escherichia coli B (SM-R) 3. 13 I Serratz'a marcescens ATCC 9986...- 25.0 I Aerabacler aerogenes ATCC 7256 6. 25 I Vibrio n 13-3-6 12. 5 I Mycubacterium 607 (SM-R)... 6. 25 I Sacchcromyces cerem'siae. 100 II Candida alblcrms IAM 4888 50. 0 II Cryptacoccus neoforma'ns IAM 4.514-- 25. 0 II Aspergillus nigcr 50. 0 II Penicillium chrysapenum FAT 917. 100. 0 II No'rn.SMR=Streptomycin resistant strain. Medium I=Bouillon. Medium II=Sahouraud's medium containing 2.0% glucose.

As is clear from the above table, the antibiotic S 15-1 has wide antimicrobial power or potency against grampositive, gram-negative and acid fast bacteria and, when increased in concentration, against molds and yeasts.

Furthermore, in a Newcastle disease virus growth-inhibiting test carried out in the tissue culture of chickembryo cell, using a culture medium incorporated with 100 lg/ml. of streptomycin and 100 U/ml. of penicillin, the incorporation of 100 ,ug./m1. of the antibiotic S 15-1 results in the complete growth inhibition of said virus. Further tests are conducted in such a manner that drinking water in which a given amount of S 15-1 is dissolved is fed every day, to find that chickens are prevented from infection or those which have already been suflfered from disease do not become more serious but rather not serious.

Amount of S 15-1 added to drinking water -y/ml. Remarks 50 Seasons when Newcastle disease likely goes on.- 100 Just when some chickens fallen ill are found. To chickens already fallen ill. 500 In addition, an aqueous solution containing 1000 'y/ml. of S 15-1 is dropped in eyes and nose or sprayed every day.

The toxicity of the antibiotic S 15-1 is such that a mouse does not die even when intravenously injected with 250 mg./ml. of the antibiotic.

The present invention is illustrated in further detail below with reference to examples, but it is needless to say that the examples are illustrative and various modifications are possible within the scope of the invention. Percent is referred to by weight per volume, unless otherwise defined.

EXAMPLE 1 Streptomyces grz'seocarneus S 15-1 (FERM-P No. 706; NRRL-5311) was inoculated in 40 liters of a liquid medium (pH 7.0) containing 3.0% of starch, 1.0% of polypeptone, 0.4% of sodium nitrate, 0.2% of potassium secondary phosphate, 0.1% of potassium chloride, 0.1% of magnesium sulfate and 0.002% of ferrous sulfate, and aerobically cultured with shaking in a jar fermentor at 28 C. for 50 hours. The culture liquor was filtered at pH 6.0 to obtain 30 liters of a filtrate (production unit 120 mcg./ml.).

The culture filtrate was passed at pH 7.0 through a column packed with 2 liters of Amberlite IRC-SO (H- form), washed with water and then eluted with 0.1 N- hydrochloric acid. The active fraction (3.0 liters) was neutralized with Amberlite IR-45 (OH-form), concentrated and then freeze-dried. The resulting crude powder was extracted with 100 ml. of methanol, and the extract was charged with 700 ml. of acetone to form a white precipitate (3.5 g.). 1.0 gram of this precipitate was purified according to cellulose column chromatography using a column packed with 250 g. of cellulose powder. As the developer was used a solvent comprising n-propanol, pyridine, acetic acid and water in a ratio of 15 :10:3:l2, and the eluted active fractions were collected. The fraction mixture was concentrated at a low temperature (30 C.) to remove the solvent, and the resulting aqueous layer was freeze-dried to form a moisture absorptive powder. This powder was dissolved in water, passed through Amberlite IR-45 (OH-form) to remove the acetic acid completely,

neutralized with hydrochloric acid and then subjected to EXAMPLE 2 Streptomyces griseocarneus S 15-1 (PERM-P No. 706; NRRL-5311) was inoculated in 20 liters of a liquid medium (pH 7.0) containing 3.0% of dextrin, 3.0% of corn steep liquor, 0.4% of sodium nitrate, 0.2% of potassium secondary phosphate, 0.1% of potassium chloride and 0.1% of magnesium sulfate, and aerobically cultured with shaking in a jar fermentor at 27 C. for 50 hours. The culture liquor was filtered at pH 6.0 to obtain 15 liters of a filtrate (production unit 60 mcg./ml.).

The culture filtrate was adsorbed on a column (1.0 liter) of Amberlite IRC-50 (H-form), washed with water and then eluted with 0.1 N-hydrochloric acid. The active fraction (1.5 liters) was neutralized with a saturated caustic soda solution, and then desalted and purified according to active carbon chromatography using a column packed with a mixture comprising g. of active carbon and 7 150 g. of Celite-545. After adsorption,-the column was washed with water and eluted with 50% (weight/weight) methanol and 0.1% (weight/weight) :hydrochloric acidcontaining 50% (weight/ weight) methanol, and the eluted active fractions were collected. The fraction mixture was concentrated at a low temperature (30 C.) 'to remove the methanol, and the resulting aqueous solution was freeze-dried to form 300 mg. of a crude powder. This powder was purified according to cellulose column chromatography using a column packed with 100 g. of cellulose. As the developer was used a solvent comprising n-butanol, acetic acid and Water in a ratio of 2: 1: 1, and the eluted active fractions were collected. The active fraction mixture was concentrated at a low temperature (30 C.) to remove the solvent, and the resulting aqueous layer was freeze-dried to form a moisture absorptive powder. This powder was dissolved in water to a pH of 7.0, passed through a column packed with 30 g. of active carbon to remove the acetic acid, washed with water and then eluted with a 0.1% (weight/weight) hydrochloric acidcontaining 80% (Weight/ weight) methanol, and the eluted active fractions were collected. The active fraction mixture was concentrated at a low temperature (30 C.) to remove the methanol, and the aqueous layer was freezedried to obtain 100 mg. (titer 1,000 mcg./ml.) of the desired substance in the form of a white powder.

What we claim is:

1. Antibiotic S 15-1, active against gram positive and negative and acid fast bacteria, having the molecular formula C H N O the infrared absorption spectrum Q8 as shown in FIG. 2', being further characterized-as'decomposing at 161 to 162' C. to develop 'ayellow' color, the said antibiotic as the free base or hydrochloride beingso'lilble'inwater and'rnethaiiol and'in'soluble in acetonefethyl acetate and chloroform, the o'ptical rotation of the hydrochloride in aqueous solution" beirigfdhf, '-'26-=, exhibiting positive colorreactions in the ninhydr'in, Feli'lings, Els'on- Morgans; Molisch, sil'ver' mirror, -anthr'on'e; "Sak'aguchis Maltol tests and negative reactions in the biure't andorcin- 01 tests, having a molecular weight of 49 6pand exhibiting R, values in paper chromatography, as follows: i.

solution .1;

References Cited l l Miller, The Pfizer Handbook of Microbial- Metabolites,

McGraw-Hill Book Co., Inc.,. New-York, N.Y., 1961 pp. 38-39.

JEROME D. GOLDBERG, Primary Examiner-*- 

